1. Field of the Invention
The present invention relates to the field of the manufacture and delivery of antisense oligonucleotides. In particular, the present invention relates to antisense oligoribonucleotides conjugated to a carrier agent that enhances membrane permeability and stability of the antisense oligoribonucleotide constructs. Particularly preferred carrier agents include 2,4-dinitrophenyl-(DNP) and 3-fluoro-4,6-dinitrophenyl-(FDNP) groups coupled at the 2'-O position of the oligoribonucleotide. More particularly, the present invention relates to anti-viral and anti-cancer antisense oligoribonucleotide constructs and their methods of use to inhibit gene expression and phenotypic changes in cells and in animals with viral or cancerous diseases. Even more particularly, the present invention relates to methods for treating animals with viral infections and cancers.
2. Background of the Technology
Antisense oligonucleotides are nucleotide, or nucleotide analogues, whose sequence is complementary to a predetermined segment of RNA, either freshly transcribed RNA or messenger RNA (mRNA). Typically, sequences of the antisense oligonucleotides are chosen so as to be complementary to a critical sequence in a gene so that if the gene is hybridized to the complementary antisense sequence, the gene cannot be expressed or is subjected to enzymatic degradation. See Stec et al. U.S. Pat. No. 5,151,510, the disclosure of which is hereby incorporated by reference in its entirety. A huge number of diseases conceivably can be mitigated, controlled, regulated, or prevented through the disabling of gene expression that antisense therapeutics allow. Thus, antisense oligonucleotides offer an incredible opportunity for the treatment of a large number of diseases.
For example, the use of antisense oligonucleotides has been proposed for the treatment of a broad range of viral infections. See Matsukura et al. Proc. Nat. Acad. Sci. U.S.A. 86:4244-4448 (1989), the disclosure of which is hereby incorporated by reference in its entirety.
Further, cancer is an area ripe with opportunities for antisense approaches. See WO 92/20348 the disclosure of which is hereby incorporated by reference in its entirety. Such application relates to antisense oligonucleotides to c-myb proto-oncogene in the treatment of colorectal cancer. See also Ensoli et al. WO 94/29,444 the disclosure of which is hereby incorporated by reference in its entirety. Such application relates to antisense oligonucleotides to basic fibroblast growth factor (bFGF) in the treatment of Kaposi's sarcoma. See further Bayever et al. Antisense Res. and Dev. 2:109-110 (1992) the disclosure of which is hereby incorporated by reference in its entirety. Such article discusses antisense oligoribodinucleotides to p53 for the treatment of acute myeloblastic leukemia (AML).
However, antisense oligonucleotides, while appearing to be exceptionally effective in certain tests, appear to suffer from several problems. It is postulated that the major limitations on antisense oligonucleotides for use in therapeutic applications is their apparent limited membrane permeability and high susceptibility to enzymatic degradation, both within and without cells. These two factors combine to limit both the concentration and the duration (half-life) of the oligonucleotides within cells. With such limited concentration and half-life of the oligonucleotides within the cells, it appears as though much of the therapeutic potential of the antisense oligonucleotides is lost.
Thus, researchers have expended considerable effort to overcome the problems of limited membrane permeability and rapid enzymatic degradation of antisense oligonucleotides. To this end, researchers have utilized a variety of techniques designed to increase membrane permeability and mitigate the enzymatic degradation of antisense oligonucleotides.
A detailed discussion of oligonucleotide design and synthesis is presented in Uhlmann et al. Chemical Renews 90:543-584 (1990), the disclosure of which is hereby incorporated by reference in its entirety.
A preferred approach that has been used to enhance membrane permeability and stability of oligonucleotides is the use of alkyl-for-O substituted and S-for-O substituted nucleotide analogues. In connection with the alkyl substituted oligonucleotides, one of the phosphate oxygen atoms that is not involved with the bridge between nucleotides is substituted with an alkyl group (particularly, methyl or ethyl). Similarly, in the S-substituted oligonucleotides (phosphorothioates), one of the phosphate oxygen atoms that is not involved in the bridge is substituted with a sulfur. In the alkyl substituted oligonucleotides, a negatively charged oxygen is replaced with a neutral and sterically undemanding alkyl group (particularly methyl). With S-substituted oligonucleotides, the negative charge on the non-bridge oxygen atoms is shared asymmetrically and located primarily on the sulfur. While, with each type of substituted oligonucleotide, membrane permeability and stability appear to be enhanced, two stereoisomers are prepared that require separation following synthesis or use of non-stereoselective synthetic methods. In S-substituted oligonucleotides, this problem can be minimized through the substitution of both of the non-bridge oxygen atoms with sulfur (phosphorodithioates). Further, each type of these substituted oligonucleotides appear to suffer from a decreased hybridization affinity with RNA in cells.
While inhibition of mRNA translation is possible utilizing either antisense oligoribonucleotides or oligodeoxyribonucleotides, free oligoribonucleotides are more susceptible to enzymatic attack by ribonucleases than oligodeoxyribonucleotides. Hence, oligodeoxyribonucleotides have generally been preferred because, upon hybridization with particular mRNA, the resulting DNA-RNA hybrid duplex is a substrate for RNase H, which specifically attacks the RNA portion of DNA-RNA hybrid at the free 2'-OH. Degradation of the mRNA strand of the duplex releases the antisense oligodeoxynucleotide strand for hybridization with additional messages from the gene.
With this problem in mind, a variety of other modified oligonucleotides have been synthesized to overcome this problem. See Uhlmann et al., supra. 2'-O-methyloligoribonucleotides have been synthesized and reportedly are completely resistant to RNA- and DNA-specific nucleases. See Sproat et al. Nucleic Acids Res. 17:3373 (1989), the disclosure of which is hereby incorporated by reference in its entirety. Less specific nucleases, however, cleave the 2'-O-methyloligoribonucleotides with varying efficiencies. Further, the same group reported the synthesis of other, larger, 2'-O-allyl-substituted oligoribonucleotides. See Iribarren et al. Proc. Nat. Acad. Sci. U.S.A., 87:7747 (1990), the disclosure of which is hereby incorporated by reference in its entirety. The paper reports that 2'-O-(2-propylene)-oligoribonucleotides are more stable than 2'-O-methyloligoribonucleotides and show improved specific binding. A branched, five carbon allyl substituted oligoribonucleotide (2'-O-(3,3-dimethyl-2-butene)-oligoribonucleotide) also substantially improved the resistance of the oligonucleotide to nuclease digestion. However, such oligonucleotide showed a substantially reduced hybridization with complementary RNA sequences.
In addition to the approaches described above for enhancing permeability and stability of oligonucleotides, a variety of groups have worked on incorporating oligonucleotides into specific carriers or binding oligonucleotides to specific or nonspecific delivery agents. For example, liposomal delivery of oligonucleotides appears to hold great promise. Liposomes are microscopic vesicles composed of mono- or multi-lamellar lipid bilayers which enter cells by phagocytosis or endocytosis. In addition to liposomes, pol-L-lysine has been used as a delivery agent to enhance interaction with cells. Other possible strategies are discussed by Uhlmann et al., supra. Nevertheless, there remains a need in the antisense oligonucleotide art for an effective approach to enhance the delivery of oligonucleotides. Further, there remains a need in the art for an effective approach to mitigate enzymatic degradation of oligonucleotides. Moreover, it would be advantageous if such benefits could be achieved without a substantial reduction in the hybridization affinity of antisense oligonucleotides to cellular RNA.